The main purpose of this project is to define the interrelationship between specific autocrine regulatory molecules and calcium homeostatic pathways in a mineralizing chondrocyte phenotype. As growth plate chondrocytes mature through a proliferative and hypertrophic phenotype, steady state intracellular calcium levels progressively increase. Growth factors and hormones appear to play a key role in the maturation process. What is not known, however, is how, or if, the regulatory molecules influence intracellular calcium homeostasis. Furthermore, preliminary evidence suggests that pH may also participate in chondrocyte phenotypic regulation. We have preliminary evidence which indicates that only three of the nearly 20 recognized cartilage target molecules (bFGF, PTH and 1,25(OH)2D3) can influence, 1) long term intracellular calcium concentrations (steady state levels), 2) short term intracellular calcium concentrations (transients) and 3) phenotypic characteristics of maturing chondrocytes. Interestingly, only one of these molecules, bFGF, is believed to be an autocrine cartilage regulator. What cannot be determined from our work (or from the literature) is if the ionic effects of these agents are the cause of, or the result of, the maturation of the cells. Also, it is unknown how other growth factors (such as TGF beta 1, 2, 3, acidic FGF and PTHrP) interact with these molecules to modulate maturation or cytosolic free calcium. In order to explore these important developmental questions we need 1) an appropriate cell isolation model in which the cells can be separated into maturationally distinct sub phenotypes, 2) to define the conditions under which intracellular calcium concentrations can be accurately monitored, 3) to exploit our expertise for the assay and study of chondrocyte targeted growth factors and hormones and 4) to manipulate the intracellular calcium pathways independently of the growth factors and hormones. Along with these objectives we will use fluorescence digital imaging for single cell calcium measurements and quantitative in situ hybridization techniques to validate our findings. Our progress toward these goals has been steady and thus, we believe it is now possible to answer some of the fundamental questions regarding chondrocyte development.